消化文章

—Original—

Histological Examination of the Relationship between Respiratory Disorders and Repetitive Microaspiration Using a Rat Gastro-Duodenal Contents Reflux Model

Keisuke OUE1, 2), Ken-ichi MUKAISHO1), Tomoki HIGO1, 2), Yoshio ARAKI1), Masanori NISHIKAWA2), Takanori HATTORI1),

Gaku YAMAMOTO2), and Hiroyuki SUGIHARA1)

Departments of 1)Pathology and 2)Oral and Maxillofacial Surgery, Shiga University of

Medical Science, Seta-tsukinowa-cho, Otsu, Shiga 520-2192, Japan

Abstract: Microaspiration due to gastroesophageal reflux (GER) has been suggested as a factor contributing to the development and exacerbation of several respiratory disorders. To explore the relationship between GER and respiratory disorders, we histologically examined the bilateral lungs of a rat gastroduodenal contents reflux model, which was previously used to investigate the histogenesis of Barrett’s esophagus and esophageal carcinoma. GER was surgically induced in male Wistar rats. The bilateral lungs of the reflux rats were examined with hematoxylin and eosin (HE), PAS-Alcian blue, and Azan staining at 10 and 20 weeks after surgery. Immunohistochemical staining of CD68 and α-SMA was also performed. Aspiration pneumonia with severe peribronchiolar neutrophilic and lymphocytic infiltrates, goblet cell hyperplasia, prominence of blood vessels, and increased thickness of the smooth muscle layer were detected. Bronchiolitis obliterans (BO)-like lesions comprising granulation tissue with macrophages, spindle cells, and multinucleated giant cells in the lumen of respiratory bronchioles were observed in the bilateral lungs of the reflux animals. These findings suggest that the severe inflammation and the BO-like lesions may play a role in exacerbation of the forced expiratory volume in 1 second (FEV 1) in human cases. In conclusion, we speculate that repetitive microaspiration due to GER may contribute to the exacerbation of various respiratory diseases, particularly asthma and chronic obstructive pulmonary disease (COPD), and the development of BO syndrome following lung transplantation. The reflux model is a good tool for examining the causal relationships between GER and respiratory disorders.

Key words: bronchiolitis obliterans, gastro-duodenal reflux, microaspiration, rat reflux model, respiratory disorder

(Received 12 August 2010 / Accepted 25 November 2010)

Address corresponding: K. Mukaisho, Department of Pathology, Shiga University of Medical Science, Seta-tsukinowa-cho, Otsu, Shiga 520-2192, Japan

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Introductiona common disorder in the USA and Western Europe in Gastroesophageal reflux disease (GERD) has become recent decades [16]. The manifestations can be divided into esophageal and extraesophageal syndromes, which affect various tissues and organ systems in addition to the esophagus [28, 41]. Among the various extraesophageal -GERD and cough, laryngitis, asthma, and dental erosion manifestations, causal relationships between have been strongly suggested in the Montreal definition [42]. In addition, gastroesophageal reflux (GER) accompanied by regurgitation and microaspiration has been -suggested as a contributory factor in several respiratory disorders. Idiopathic pulmonary fibrosis, cystic fibrosis, connective tissue disease, and obstructive lung disease have all been reported to be associated with GER [19, 39, 42]. Moreover, GER has been shown to worsen asthma through esophagobronchial reflex, and to heighten bronchial reactivity and microaspiration [2, 14, 20]. -GER has also been reported to be accompanied by neu trophilic airway inflammation [10] and it has been sug-gested that patients with chronic obstructive pulmonary -disease (COPD) along with GERD symptoms are more likely to experience exacerbations than those lacking these symptoms. GERD may also increase the tracheobronchial aspiration of gastric juice directly and/or dis-turb the clearance of swallowed contents from the phar-ynx exacerbations. Therefore, GER may act as a confoundto the esophagus indirectly, leading to frequent -ing factor in COPD exacerbations through mechanisms -similar to those seen in asthma and/or by increasing airway inflammation [38].

monly observed in lung transplant patients in recent Microaspiration due to GER has been most com-years. Lung transplantation has become a therapeutic option for appropriately selected patients with end-stage lung diseases in the last 20 years. However, successful approaches for managing chronic rejection of pulmonary allografts have not yet been developed. Long-term survival of patients with pulmonary allografts is currently -hindered by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection that is unique to lung transplantation. Clinical studies suggest that both immune- -and non-immune-mediated factors contribute to BOS

development. High GER levels with resultant aspiration have been implicated as a non-immune risk factor for BOS development following lung transplantation [15, 22, 31, 34, 35, 43]. GER is promoted after lung transplantation probably because of the potential for iatro-genic vagal nerve injury during lung transplantation and -due to the use of immunosuppressive drugs such as calcineurin inhibitors, cyclosporine and tacrolimus that -prolong gastric emptying [5, 15, 35]. In addition, bile acids in bronchoalveolar lavage fluid at three months after transplant were found to be associated with BOS development in a time- and dose-dependent manner [17]. These findings suggest that bile acids, not gastric juice in reflux contents, play an important role in BOS development following lung transplantation.

-relationships between GER and respiratory disorders, In order to establish experimental evidence for causal we examined both lungs of the reflux model, which had previously been used to investigate the histogenesis of Barrett’s esophagus and esophageal carcinoma [11, 12] and was recently used to examine the mechanism of development of extraesophageal syndromes including laryngitis and dental erosion [26, 30].

Materials and Methodsfor All procedures complied with the ethical guidelines laboratory animals at Shiga University of Medical Scianimal experimentation and the care and use of ence, Japan.

-Animal models

male Wistar rats (CLEA, Tokyo, Japan) according to a Gastro-duodenal reflux was induced in eight-week-old previously reported procedure [11, 12]. Shortly afterwards, the esophagogastric junction was transected, the -distal cut end was closed, and the proximal cut end was anastomosed end-to-side to the upper jejunum, approximately 2 cm distal to the origin. After the esophagoje-junostomy, a serosal suture (interrupted 7-0 nylon) was -placed between the esophagus and jejunum to support the afferent loop side of the anastomosis. As a result, the serosal suture allowed ingested food to easily enter the efferent loop and prevented food from entering the afferent loop. The gastro-duodenal contents flowed through

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the esophagojejunal stoma and back into the esophagus [11, 12] (Fig. 1). Sham-operated rats, which underwent a laparotomy with blunt manipulation of the abdominal contents under diethyl ether anesthesia, were used as con-trol animals. The reflthanized with an overdose of diethyl ether at postoperative ux animals that survived were eu-week 10 (n=7) or 20 (n=7). Sham-operated control rats were euthanized at similar time points (n=7 per group).Histological examination

and fiResected bilateral lung tissue samples were weighed for 4 h and embedded in paraffi xed in 10% formalin in phosphate-buffered saline tions were made and stained with hematoxylin and eosin n. Four-micrometer sec-(HE). PAS-Alcian blue staining for detection of goblet cell metaplasia and Azan staining for fiperformed on the lung samples. The degree of positive brosis were also cells for PAS-Alcian blue staining was recorded as weak (0), mild (1+), moderate (2+), or strong (3+).

Immunohistochemical stainings of CD68 and αmouse monoclonal antibodies: CD68 (a marker for mac-Immunohistochemical staining was performed with -SMArophages, Clone:ED1, 1:500, AbD Serotec, Oxford, UK) and actin, Clone:ASM-1, 1:200, Progen Biotechnik, Heidel-α-SMA (a marker for α-smooth-muscle isoform of berg, Germany).covery XT Automated IHC Stainer using the Ventana The staining was performed on a Dis-DABMap detection kit (catalog No. 760-124, Ventana Medical Systems, Tucson, AZ, USA). Each step of the Ventana DABMap detection kit procedure was optimized on the Discovery XT instrument and preset. Antigen retrieval of tissue sections was performed with enzyme for CD68 and with heat for counterstained with haematoxylin. Slides of the negative α-SMA. The sections were control without the primary antibody and those of the positive control were processed in parallel.

Thickness of smooth muscle layer of bronchioles

was quantifiThe thickness of the bronchiolar smooth muscle layer positive layer at 4 points in randomly selected bron- ed by measuring the thickness of α-SMA chiolar cross sections of the smallest diameters, 250–500 µrecorded.

m. The mean of the measurements in each groups was Fig. 1. Gastro-duodenal contents reflsingle serosal suture between the esophagus ux model. A

and jejunum was added after the esophago-jejunostomy. B, Bile duct; E, esophagus; F, fore stomach; G, glandular stomach; D, duodenum; J, jejunum.

pH analysis of the esophageal and gastric contents tric contents was estimated using a Compact pH meter Following euthanasia, pH of the esophageal and gas-(Horiba, Kyoto, Japan).

Statistical analysis

tric contents, and the thickness of the smooth muscle Absolute lung weights, the pH of esophageal and gas-layer of bronchioles are expressed as mean ± SE. Stu-dent’s P<0.05 was considered statistically signifit-test or Welch’s t-test were used for comparisons. cant.

ResultspH of the esophageal and gastric contents3.75 ± 0.19 after 10 weeks and 4.11 ± 0.34 after 20 weeks The pH of the gastric contents in the refl ux rats was compared to control rat values of 4.06 ± 0.30 after 10 weeks and 4.32 ± 0.49 after 20 weeks. The pH of the esophageal contents in the reflafter 10 weeks and 6.48 ± 0.08 after 20 weeks. How- ux rats was 7.07 ± 0.11 ever, no comparable pH measurements were made in control rats at 10 and 20 weeks due to the small volume of esophageal contents in these animals. There were no signifibetween the groups (Table 1).

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Table 1. Esophageal and gastric content pH

Gastric pH Esophageal pH

Control (10 w) 4.06 ± 0.30 7.29 ± 0.06

Reflux model (10 w)

3.75 ± 0.19

n.d.

Control (20 w) 4.32 ± 0.49 6.40 ± 0.08

Reflux model (20 w)

4.11 ± 0.34

n.d.

The data are shown as the mean ± SE. n.d., not determined; due to very little fluid in the esophagus of control animals.

Table 2. Lung weights

R. lung (g) L. lung (g)

Control (10 w) 0.812 ± 0.03 0.440 ± 0.02

Reflux model (10 w)

0.822 ± 0.05 0.466 ± 0.04

Control (20 w) 0.8 ± 0.03 0.467 ± 0.01

Reflux model (20 w)

0.854 ± 0.110.536 ± 0.09

The data are shown as the mean ± SE.

Bilateral absolute lung weights

Bilateral absolute lung weights are summarized in Table 2. At both 10 and 20 weeks, no significant increase of bilateral absolute lung weights from both control rats and reflux rats were noted. However, the left lung weights from reflux rats at 20 weeks were slightly high-er than those of the controls. The lung weights appeared to correlate with the degree of inflammatory cell infil-trates in the lungs of the reflux animals.

Macroscopic and under-loupe findings

Both lungs of the reflux animals were partially con-solidated compared to those of the control animals at 10 and 20 weeks following surgery (Fig. 2). HE-stained sections showed several areas of densely stained inflam-matory cell infiltrates, and atelectasis was detected in the bilateral lung fields of the reflux animals (Fig. 3). These areas grew in size in a time-dependent manner.Microscopic findings

Low-power views of both lungs showed partial con-solidation in areas surrounding the bronchi and bronchi-oles of the reflux animals (Fig. 4a). High-power views of the lung alveolar spaces of reflux animals showed partly severe lymphocytic and neutrophilic infiltrates, macrophages, and multinucleated giant cells within the alveolar space (Fig. 4b and 4c). In addition, bile acids and food contents were occasionally found within multi-nucleated giant cells. In severe cases, the bronchioles were partially plugged with neutrophilic exudates (Fig. 4c and 4d). Neither congestion nor pulmonary edema was detected in any animal.

A markedly increased number of goblet cells positive for PAS-Alcian blue and an increased number of peri-bronchiolar blood vessels were detected in the reflux animals compared to the control animals (Fig. 5a–d). PAS-Alcian blue staining showed a time-dependent in-crease in the number of goblet cells. Goblet cells ac-counted for a significantly greater number of bronchial and bronchiolar epithelial cells at 20 weeks after surgery than at 10 weeks. We detected strong (3+) proliferation of goblet cells, i.e., goblet cell hyperplasia, in the bron-chial and bronchiolar epithelium in three of seven spec-imens at 10 weeks and all seven specimens at 20 weeks in the left lung, and five of seven specimens at 10 weeks and all seven specimens at 20 weeks in the right lung (Table 3). The thickness of the smooth muscle layer of the bronchiole in the bilateral lungs of the reflux animals was significantly greater than that of the control animals at 10 and 20 weeks after surgery (Table 4). Bronchioles of 250–500 µm representatives of the smallest diameters in the reflux and control animals are shown in Fig. 6. We also observed that many reflux animals had BO-like lesions, characterized by peribronchiolar lymphocytic infiltrates and luminal narrowing of the respiratory bron-chioles due to polypoid granulation tissue composed of numerous macrophages positive for CD68, multinucle-ated giant cells, and spindle cells (Fig. 7a and 7b). Fi-brous connective tissue, partially positive for Azan stain, was observed in the granulation tissue (Fig. 7c). BO-like

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Fig. 2. Macroscopic views of lungs at 20 weeks postoperatively. a) sham-operat-ed animal, b) refl ux animal. Gross fi gure shows that the color of bilateral lungs in the refl ux animals is markedly red and bilateral lungs in the refl ux animals are partly consolidated compared to those of the control animals.

Fig. 3. Loupe views of HE sections. a–c, left

lung; d–f, right lung; a and d, control animal at 20 weeks; b and e, refl ux animal at 10 weeks postoperatively; c and f, re-fl ux animal at 20 weeks postoperatively. Several parts with dense infl ammatory cell infi ltrates were detected in bilateral lung fi elds. These infi ltrates were more severe at 20 weeks than at 10 weeks after operation.

Fig. 4. Microscopic fi ndings in HE sections from

the refl ux animals at 20 weeks. a, Low-power view; b–d, high-power view. a) Severe lymphocytic and neutrophilic in-fi ltrates, macrophages, and multinucle-ated giant cells were detected. b) Many multinucleated giant cells were found in the alveolar spaces. c and d) Many neu-trophilic infi ltrates were found within the alveolar spaces and in the small airway.

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Fig. 5. Goblet hyperplasia and an increased number of blood vessels developed in the refl ux animals at

20 weeks. a and d, HE staining; b and e, PAS-Alcian blue staining; c, and f, immunohistochemical staining of α-SMA; a–c, control animal; d–f, refl ux animal. Marked goblet cell hyperplasia posi-tive for PAS-Alcian blue was observed in the refl ux animals compared to the control animals (a, b, d, e). Not only bronchiolar smooth muscle layer but also peribronchiolar blood vessels were positive for α-SMA (c and f). An increased number of blood vessels, and infl ammatory cell infi l-trates were also found in the refl ux animals (a, c, d, f).

Fig. 6. Comparison of the thickness of the bronchiolar smooth muscle layer of refl ux

animals and controls at 20 weeks after surgery. a) Control animal, b) Refl ux model. We measured the thickness at 4 points in each cross-section of the small-est diameter bronchioles, 250–500 µm. The thickness of the smooth muscle layer in the refl ux animals was signifi cantly greater than in the controls.

Fig. 7. Bronchiolitis obliterans-like lesions developed in the refl ux animals at 20 weeks. a) HE staining, b) CD68, c) Azan staining. Bronchiolitis obliterans-like lesions comprising granulation tissue with macrophages positive for CD68, spindle cells, and a multinucle-ated giant cell in the lumen of respiratory bronchioles were observed in the refl ux animals (a and b). Partially fi brous connective tissue positive for Azan staining was noted in the granulation tissue (c).

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Table 3. Incidence of bronchiolitis obliterans-like lesion and goblet cell metaplasia in

control and reflux animals

Control (10 w) Control (20 w)

Reflux model (10 w) Reflux model (20 w)

Bronchiolitis obliterans-like lesion

L. lung 0/7 0/7 4/7 7/7

R. lung 0/7 0/7 2/7 7/7

Goblet cell metaplasia (3+)L. lung 0/7 0/7 3/7 7/7

R. lung0/70/75/76/7

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Table 4. Thickness of smooth muscle layer

Control (10 w) Control (20 w)

Reflux model (10 w) Reflux model (20 w)

L. lung Diameter of bronchiole

R. lung Thickness of smooth muscle bundles

L. lung R. lung12.7 ± 0.47* 10.9 ± 0.31** 17.5 ± 0.98* 17.3 ± 1.14**

11.7 ± 0.52*9.52 ± 0.43**21.3 ± 0.88*15.3 ± 0.58**

290.0 ± 24.2 313.7 ± 16.4 335.2 ± 16.7 295.0 ± 11.3 300.4 ± 10.0 297.5 ± 12.7 329.1 ± 21.6 321.6 ± 20.2

Data are shown as the mean ± SE. There were no significant differences among the groups in the thickness of the smooth muscle layer in the smallest diameter bronchioles examined. The mean thickness of bronchiolar smooth muscle layer was significantly greater in the reflux animals than in the control animals (* and **, P<0.0001; Student’s t-test).

lesions were detected in four of seven specimens at 10 weeks and in all seven specimens at 20 weeks in the left lung, and in two of seven specimens at 10 weeks and all seven specimens at 20 weeks in the right lung (Table 3).

DiscussionWe detected bronchopneumonia with foreign body reaction, namely aspiration pneumonia and peribron-chial atelectasis and fibrosis following purulent inflam-mation with severe peribronchiolar neutrophilic and lymphocytic infiltrates in our reflux model. Goblet cell hyperplasia, prominence of blood vessels, and increased thickness of the smooth muscle layer were also detected in association with the continuous chronic inflammatory stimulation of GER. The most important findings of the present study were BO-like lesions characterized by luminal narrowing of the respiratory bronchioles due to polypoid granulation tissue composed of numerous mac-rophages, multinucleated giant cells, and spindle cells. Based on the abovementioned results, we hypothesize that GER may play an important role in the exacerbation of various respiratory diseases, especially asthma, COPD, and BOS following lung transplantation.

In human asthma cases, GER appears to be associated with airway inflammation and has been recognized as an important causative factor in asthma attacks [21, 23]. However, the mechanism by which GER may provoke airway inflammation is doubtful. A key role seems to be played by the vagally-mediated neurogenic reflex or by the microaspirated refluxate that probably mediates airway inflammation [9, 24]. Irrespective of the active mechanism, airway inflammation in GER can be the linkage by which GER eventually exacerbates asthma. A recent study showed that GER associated with asthma is characterized by an increase in the numbers of eosino-phils and neutrophils, while GER alone presents a neu-trophilic pattern of inflammation. In addition, GER appears to exacerbate already existing oxidative stress [10]. These findings are consistent with the pattern of aspiration pneumonia with severe peribronchiolar neu-trophilic and lymphocytic infiltrates found in the present animal model. Therefore, the inflammation caused by GER can be a causative factor of exacerbation of asthma. Moreover, it has also been reported that a marked in-crease in the goblet cells in the airways is a characteris-tic feature of patients with bronchial asthma who have

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died of severe acute attacks. The discovery of goblet cell hyperplasia could be used as a marker to identify patients with bronchial asthma who are at risk of severe attacks [1]. Considering these results, the goblet cell hyperplasia observed in the present study may also play a role in worsening asthma in human cases.

chronic deterioration in respiration [3, 7]. One of the COPD exacerbations are characterized by acute and most common causes of COPD exacerbations is tracheobronchial infection. The most frequent route for -bacterial infection of the tracheobronchial tree is the silent aspiration of oropharyngeal secretions [36]. Stud-ies on the relationship between the swallowing function and GERD have revealed that subjects presenting with cough and GERD have significantly reduced laryngopharyngeal sensitivity, potentially resulting in an in-creased swallowing reflex [32]. A recent study suggested that risk of aspiration via the impairment of the -swallowing abnormalities, probably via GERD comorbidity, were associated with frequent COPD exacerba-tions [37]. Our previous study using the same rat reflux -model as the present study showed that the reflux of gastro-duodenal contents causes severe laryngitis [30]. Inflammation of the larynx must play a role in impair ment of the swallowing reflex.

-cause of death after lung transplantation; it affects 50–BOS is a major long-term complication and a leading 70% of transplant recipients [4, 6, 25]. The histological hallmark of BOS is BO [8]. BOS was originally defined as a decrease in forced expiratory volume in 1 second (FEV 1) [13]. In the present study, we detected BO-like lesions associated with repetitive microaspiration caused by GER. These lesions may play an important role in exacerbation of FEV 1 subsequent to COPD exacerbations, unlike true constrictive BO that is fused to the -bronchiolar wall and obliterates the lumen. In a recent experimental study, Li evidence consistent with BO development in lung alet al. [29] reported histological lografts after inducing chronic gastric fluid aspiration in -a rat model that utilized immunosuppression to avoid acute rejection. However, the BO-like lesions observed in the present reflux animal lungs did not result from lung transplants or cyclosporine treatments. Thus, as previously suggested by Li GER plays an important role in BO-like lesion developet al., our results imply that -

ment and that alloimmune injury following bone marrow transplantation may promote BO development.

ate in GER involves the determiation of the degree of Another issue to be investigated with regard to reflux-harm and importance of gastric juice and bile acids in the pathogenesis of BO. The refluxates of the reflux animals in the present study contained not only gastric juices but also duodenal contents including bile acids. D’Ovidio veolar lavage fluid of 120 post-transplant patients in a et al. examined bile acids in the bronchoal- cross-sectional study and found a 17% prevalence of elevated bile acid concentrations. Furthermore, the highest -onset BOS [18]. Bile aspiration secondary to duodeno-concentrations were found in patients with early GER has been associated with severe pulmonary injury. Cytotoxicity may result in disruption of cellular mem branes or alteration of cellular cationic permeability -depending on the bile acid concentration [27, 33, 40]. These findings suggest that refluxates, including bile acids, play an important role in BO pathogenesis in lung transplant patients.

foreign body reaction and peribronchial atelectasis and In summary, we detected bronchopneumonia with fibrosis in conjunction with purulent inflammation with severe infiltrates in our reflux-induced rats. We also noted gobperibronchiolar neutrophilic and lymphocytic let cells and smooth muscle hyperplasia. From these -findings, we speculate that repetitive microaspiration due to GER contributes to the exacerbation of various respiratory diseases, especially asthma and COPD. BO-like -lesions were also observed in reflux animal lungs without lung transplantation or cyclosporine treatment. These findings confirm that GER including bile acids plays an important role in the development of BO as a nonimmune-mediated factor in lung transplant patients. The -gastro-duodenal contents reflux model was originally used model is a good tool for studying the development of in studies of Barrett’s esophagus; however, the extraesophageal syndromes and respiratory disorders based on GER.

Acknowledgmentassistance.

We thank Mr. Michiharu Nasu for excellent technical RESPIRATORY DISORDERS AND MICROASPIRATION IN RATS

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